Figure 6: K⁺ efflux or hyperpolarization mediated by acetate induces inflammasome activation in primed human colonic epithelial cells.

(a) NLPR3 normalized expression with GAPDH was measured in HT29 cells incubated for 2 h with diluted faecal supernatant (FS) or with PBS by real-time PCR. Results are shown as mean±s.e.m. of duplicate of a representative experiment. (b) Caspase 1 activity was measured via a fluorometric assay measuring the cleavage of the peptide YVAD-AFC. NMC460 cells were incubated with diluted FS for 4 h followed by 2 h with PBS (FS), incubated with FS for 4 h followed by 2 h in low K⁺ medium (FS-low K⁺) or incubated with FS for 4 h followed by 2 h with 10 mM acetate (FS-acetate). Results are shown as mean±s.e.m. of triplicate of a representative experiment. (c–f) Changes in membrane potential following addition of vehicle or 10 mM acetate to NMC460 (c), HT29 (d) CHOK1 transfected with GPR43 (e) or untransfected (f). Membrane potential was measured using a FLIPR membrane potential assay kit (red) from Molecular Devices. (g) Measurement of Ca²⁺i mobilization in CHO cells stably expressing GPR43 mediated by 10 mM acetate or vehicle. Data are mean±s.e.m. of duplicates from a representative experiment. (h) Change in membrane potential following addition of vehicle or 10 mM acetate with or without 20 μM BAPTA-AM to NCM460 cells. Membrane potential was measured using a FLIPR membrane potential assay kit (red) from Molecular Devices. Data are mean±s.e.m. of duplicates from a representative experiment.